Defects in the function of retinal microvessels are responsible for much of the retinal injury that occurs in diabetes. To study properties of retinal capillaries at a cellular level, we isolate microvessels from bovine retina and prepare primary cultures of endothelial cells and pericytes. The cells are separated from each other by density centrifugation. Contamination is minimized by use of selective growth media and attachment substrate. Enhanced permeability of retinal microvessels is a prominent reaction in diabetes. We propose to study the in-vitro barrier created by retinal endothelial cells grown on nylon mesh. Using scanning electron microscopy and freeze fracture analysis we find that the cells form a continuous monolayer and are joined together by numerous tight junctions. The ultrastructure, membrane electrophysiology, and transport properties of the monolayers will be determined. The effect of exposure to high concentrations of glucose will be measured. Because capillary cells are damaged in diabetic retinopathy, the synthesis of sorbitol by pericytes and endothelial cells in culture will be measured and correlated with signs of cell injury. In preliminary experiments we found that pericytes but not endothelial cells accumulate sorbitol after exposure to high concentrations of glucose. We will determine if this difference could be responsible for the selective vulnerability of retinal pericytes. Finally, since basement membrane thickening is a consistent finding in diabetic microangiopathy, the effect of high concentrations of glucose on the production of basement membrane proteins by the two cell types will be assayed. The long-term objective of this investigation is to characterize the cellular properties of retinal microvessels that underlie their vulnerability in diabetes mellitus.
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